Evaluation of Mycobacterium tuberculosis genes involved in resistance to killing by human macrophages

A coinfection assay was developed to examine Mycobacterium tuberculosis gen es suspected to be involved in resistance to killing by human macrophages. THP-1 macrophages were infected with a mixture of equal numbers of recombin ant Mycobacterium smegmatis LR222 bacteria expressing an M. tuberculosis ge ne and wild-type M. smegmatis LR222 bacteria expressing the xylE gene. At v arious times after infection, the infected macrophages were lysed and the b acteria were plated. The resulting colonies were sprayed with catechol to d etermine the number of recombinant colonies and the number of xylE-expressi ng colonies. M. smegmatis bacteria expressing the M. tuberculosis glutamine synthetase A (glnA) gene or open reading frame Rv2962c or Rv2958c demonstr ated significantly increased survival rates in THP-1 macrophages relative t o those of xylE-expressing bacteria. M. smegmatis bacteria expressing M. tu berculosis genes for phospholipase C (plcA and plcB) or for high temperatur e requirement A (htrA) did not.